The recent rise in oil prices has caused an increased interest in alternative fuels such as biofuel. Also, butanol, a gasoline alternative having excellent physical properties compared to ethanol, is of increasing interest, and thus Clostridium sp. strains which produce solvents such as butanol as metabolites are also of increasing interest.
It is known that the microorganisms of the genus Clostridium are gram-positive, strictly anaerobic, endospore-forming bacteria and mostly produce acetic acid and butyric acid as fermentation products. Among them, some strains cause acetone-butanol-ethanol fermentation (hereinafter referred to as ABE fermentation) which produces acetone, butanol and ethanol in addition to the above organic acids.
Indeed, in the early 20th century, Clostridium acetobutylicum that is one of such strains was used to produce acetone and butanol in large amounts. This mass production was continued up to the 1960s and 1970s, but was discontinued except for some countries, because acetone and butanol produced from crude oils were inexpensive due to the development of chemical processes and it was difficult to supply substrates.
The production of biobutanol which has been performed to date using Clostridium sp. strains entails the following problems. First, it shows significantly low yield and productivity compared to the production of bioethanol based on yeast. Second, the Clostridium sp. strains produce, in addition to butanol that is highly valuable as biofuel, byproducts, including acetone, acetic acid and butyric acid, which increase their separation costs.
In Clostridium sp. strains that produce solvents, the productions of organic acids occurs at the exponential growth phase, like general microbial fermentation. It is referred to as the acidogenic phase. As the stationary phase approaches, the metabolism of cells shifts to the solventogenic phase in which organic acids are reassimilated and solvents such as acetone (or isopropanol), butanol and ethanol are produced. This can be interpreted as follows. As the stationary phase approaches, the pH decreases, and thus the concentration of non-dissociated organic acids increases. Among these acids, non-dissociated butyric acid shows high cytotoxicity. Through this reassimilation of organic acid and conversion into solvents, cells gain time to form endospores that can survive for a long period of time in a severe environment.
In wild-type strains, acetone, butanol and ethanol are produced at a mass ratio of about 3:6:1 after fermentation, and trance amounts of acetate, butyrate and acetoin are also produced. It is known that when glucose is used as a substrate, butanol is produced with a mass yield of about 20-25% at a final concentration of about 10 g/L (Lee et al., Biotechnol. Bioeng., 101(2):209-228, 2008). When such wild-type strains are used to produce butanol, there are problems in that yield and productivity are low and butanol is difficult to separate from other metabolites, thus increasing the production costs.
For this reason, in recent years, efforts have been made to make improved strains using metabolic engineering approaches, which manipulate metabolic pathways as desired, based on genetic engineering knowledge and tools. For Clostridium acetobutylicum, the pathways that produce metabolites have been known for a long time, and the genome was sequenced while genes corresponding thereto were all identified (Nolling et al., J. Bacteriol. 2001).
A metabolite that is most problematic in butanol production is acetone. If gas stripping is used for solvent separation, acetone and butanol are separated as mixtures because they are all easily evaporated, unlike organic acids, and an additional separation process is required. Acetoacetyl-CoA is converted to acetone by CoA transferase and acetoacetate decarboxylase. These enzymes are expressed by the genes ctfAB and adc, respectively. Thus, the concentration of acetone in solvents can be reduced by deleting one or more of these genes. According to a recent report, it was found that the deletion of adc can reduce the concentration of acetone can indeed the ratio of acetone (Jiang et al., Metab. Eng., 11(4-5):284-291, 2009).
Also, in the case of Clostridium, there is an example in which pta, a gene expressing phosphotransacetylase that is an enzyme of the acetate-producing pathway, and buk, a gene expressing butyrate kinase, were deleted by insertion of a plasmid by single crossover (Green et al., Microbiology. 142:2079-2086, 1996). However, it was reported that, when the buk gene that encodes butyrate kinase was deleted, the concentration of butanol was increased to about 16 g/l, but when the pta gene that encodes phosphotransacetylase was deleted, the concentration of butanol was 9.9 g/l, indicating that the concentration of butanol and the selectivity for butanol did not substantially increase.
WO2008/052973 discloses a strain wherein the butyrate-producing pathway and the acetate-producing pathway are blocked and a strain wherein the butyrate-producing pathway, the acetone-producing producing pathway and the acetate-producing pathway are blocked. However, it is essential to block the butyrate-producing pathway, and thus it is impossible to determine whether the ability to produce butanol is increased when the acetate-producing pathway alone is deleted. In addition, WO2008/052973 discloses deletions of various combinations of genes based on a deletion of buk or ptb, but the examples thereof show only the already known results obtained by deleting the buk gene, and this patent document discloses an example relating to deletions of various combinations of genes. In other words, this patent document generally suggests only the possible deletions of various combinations of genes based on the buk or ptb deletion without providing a scientific experimental basis, but it is impossible to determine whether these deletions contribute to increases in concentration, yield, selectivity and the like in actual butanol production.
Thus, according to reports known to date, there is no evidence that the pta deletion is helpful in increasing the concentration and yield of butanol. In addition, because there is no information on the pta deletion, it is unclear what is the expected outcome if buk is deleted in a pta-deleted mutant strain. Also, if both the buk gene involved in the production of butyrate and the pta gene involved in the production of acetic acid are deleted, it can be expected that butyrate or acetic acid will not be produced, and thus the yield of butanol will increase (WO2008/052973), but this is an incorrect expectation (see the detailed description below).
Accordingly, the present inventors have found that, when a gene encoding an enzyme that converts acetyl CoA to acetate is deleted in the microorganisms of the genus Clostridium, the selectivity and yield of butanol are increased, indicating that the ability of the microorganisms to produce butanol is increased, thereby completing the present invention.
Furthermore, the present inventors have constructed microorganisms, which produce butanol at high concentration with high yield and selectivity, by deleting the buk gene in a pta-deleted mutant strain having improved butanol selectivity and yield and amplifying aldehyde/alcohol dehydrogenase in the mutant strain, and have found that the constructed microorganisms are able to produce butanol at high concentration with high yield and high selectivity, thereby completing the present invention.
Moreover, the present inventors have constructed a strain, which produces butanol at high concentration with high yield and selectivity without substantially producing organic acid, by deleting the bukII gene, which encodes butyrate kinase, in the mutant strain in which both pta and buk were deleted and aldehyde/alcohol dehydrogenase was amplified, and have confirmed the ability of the strain to produce butanol, thereby completing the present invention.
In addition, the present inventors have constructed a strain, which produces butanol at high concentration with high yield and selectivity without substantially producing organic acid, by deleting the ctfB gene, which encodes CoA transferase (CoAT), in the mutant strain in which pta, buk and bukII were all deleted and aldehyde/alcohol dehydrogenase was amplified, and have confirmed the ability of the strain to produce butanol, thereby completing the present invention.